Synthesis of 2-Deoxybrassinosteroids Analogs with 24-nor, 22(S)-23-Dihydroxy-Type Side Chains from Hyodeoxycholic Acid

Natural brassinosteroids are widespread in the plant kingdom and it is known that they play an important role in regulating plant growth. In this study, two new brassinosteroid analogs with shorter side chains but keeping the diol function were synthesized. Thus, the synthesis of 2-deoxybrassinosteroids analogs of the 3α-hydroxy-24-nor, 22,23-dihydroxy-5α-cholestane side chain type is described. The starting material is a derivative from hyodeoxycholic acid (4), which was obtained with an overall yield of 59% following a previously reported five step route. The side chain of this intermediate was modified by oxidative decarboxylation to get a terminal olefin at the C22-C23 position (compound 20) and subsequent dihydroxylation of the olefin. The resulting epimeric mixture of 21a, 21b was separated and the absolute configuration at the C22 carbon for the main product 21a was elucidated by single crystal X-ray diffraction analysis of the benzoylated derivative 22. Finally, lactonization of 21a through a Baeyer-Villiger oxidation of triacetylated derivative 23, using CF3CO3H/CHCl3 as oxidant system, leads to lactones 24 and 25 in 35% and 14% yields, respectively. Deacetylation of these compounds leads to 2-deoxybrassinosteroids 18 and 19 in 86% and 81% yields. Full structural characterization of all synthesized compounds was achieved using their 1D, 2D NMR, and HRMS data.


Introduction
Since the discovery of brassinolide (1), a polyhydroxysteroidal hormone that regulates plant growth and development, other brassinosteroids (BRs) have been found throughout all the plant kingdom and much effort has been dedicated to the synthesis of BR analogs. Most of this work has been focused on determining the structural requirements that these compounds should possess to elicit strong biological activity [1][2][3]. For example, in Figure 1 are shown the chemical structures of 1, castasterone (2) and typhasterol (3). The latter is a natural 2-deoxybrassinosteroid that may act as important biosynthetic precursors of more active brassinosteroids [4][5][6][7]. Natural occurring BRs show a variety of structural modifications in the A/B ring, but it seems that a vicinal 22R,23R diol structural functionality in the side chain is essential for high biological activity. In recent decades, many BR analogs with structural changes on the A/B rings and/or on the side chain (shorter side chains, different oxygenated functions, spirostanic, aromatic and cyclic substituents, methyl esters, carboxylic acids) have been synthesized [8][9][10][11][12]. Surprisingly, some BR analogs with drastic structural modifications in the side chain have also shown interesting activities as plant growth regulators. Thus, the structural requirement of a side chain with a cis C-22, C-23-diol, preferentially with R, R configurations, and a C-24 methyl or ethyl substituent, seems to be contradicted by an important number of BR analogs exhibiting strong biological activities. For example, in Figure 2 are shown a series of BR analogs with 24-nor-22,23-dihydroxy-type side chains, i.e., BRs analogs with shorter side chain as compared to naturally occurring BRs. Hyodeoxycholic acid (4) has been used in the synthesis of several BRs analogs because its structure is similar to that of active BRs and it is commercially available. Thus, compounds 5-8 and 8-9 have been synthesized from 4 following different synthetic routes [13,14]. In both cases, the modification in the side chain was achieved by decarboxylation and subsequent dihydroxylation of a terminal olefin. From these compounds only 8 and 9 were evaluated as potential Natural occurring BRs show a variety of structural modifications in the A/B ring, but it seems that a vicinal 22R,23R diol structural functionality in the side chain is essential for high biological activity. In recent decades, many BR analogs with structural changes on the A/B rings and/or on the side chain (shorter side chains, different oxygenated functions, spirostanic, aromatic and cyclic substituents, methyl esters, carboxylic acids) have been synthesized [8][9][10][11][12]. Surprisingly, some BR analogs with drastic structural modifications in the side chain have also shown interesting activities as plant growth regulators. Thus, the structural requirement of a side chain with a cis C-22, C-23-diol, preferentially with R, R configurations, and a C-24 methyl or ethyl substituent, seems to be contradicted by an important number of BR analogs exhibiting strong biological activities. For example, in Figure 2 are shown a series of BR analogs with 24-nor-22,23-dihydroxy-type side chains, i.e., BRs analogs with shorter side chain as compared to naturally occurring BRs. Natural occurring BRs show a variety of structural modifications in the A/B ring, but it seems that a vicinal 22R,23R diol structural functionality in the side chain is essential for high biological activity. In recent decades, many BR analogs with structural changes on the A/B rings and/or on the side chain (shorter side chains, different oxygenated functions, spirostanic, aromatic and cyclic substituents, methyl esters, carboxylic acids) have been synthesized [8][9][10][11][12]. Surprisingly, some BR analogs with drastic structural modifications in the side chain have also shown interesting activities as plant growth regulators. Thus, the structural requirement of a side chain with a cis C-22, C-23-diol, preferentially with R, R configurations, and a C-24 methyl or ethyl substituent, seems to be contradicted by an important number of BR analogs exhibiting strong biological activities. For example, in Figure 2 are shown a series of BR analogs with 24-nor-22,23-dihydroxy-type side chains, i.e., BRs analogs with shorter side chain as compared to naturally occurring BRs. Hyodeoxycholic acid (4) has been used in the synthesis of several BRs analogs because its structure is similar to that of active BRs and it is commercially available. Thus, compounds 5-8 and 8-9 have been synthesized from 4 following different synthetic routes [13,14]. In both cases, the modification in the side chain was achieved by decarboxylation and subsequent dihydroxylation of a terminal olefin. From these compounds only 8 and 9 were evaluated as potential Hyodeoxycholic acid (4) has been used in the synthesis of several BRs analogs because its structure is similar to that of active BRs and it is commercially available. Thus, compounds 5-8 and 8-9 have been synthesized from 4 following different synthetic routes [13,14]. In both cases, the modification in the side chain was achieved by decarboxylation and subsequent dihydroxylation of a terminal olefin. From these compounds only 8 and 9 were evaluated as potential neuroinflammation inhibitors [14]. On the other hand, compounds 10-13 were synthesized from deoxycholic acid, bearing oxygenated functions in ring C, with 24-nor-22(S),23-dihydroxy side chain and cis A/B ring fusion [15], whereas analogues 14-16 were obtained from deoxycholic acid with 11-oxo-functionalized on C ring, 24-nor-22(S),23-dihydroxy and 22(S),23-diacetoxy [16]. Interestingly, compounds 10 and 13 have shown growth promoting activity in hypocotile elongation and cothyledon expansion in a radish bioassay [17].

Results
The main goal of this work was to synthesize BR analogs where the main structural change was a reduction of the side alkyl chain length, as compared to brassinolide (1), but keeping the glycol function at the C22C23 position. In the steroidal nucleus, the introduction of a glycol function at the C22C23 position via dihydroxylation with OsO4 requires the presence of a terminal double bond. In the case of hyodeoxycholic acid (4) this can be accomplished by oxidative decarboxylation using a Pb(OAc)4/Cu(OAc)2 system. This method has been proposed to obtain terminal double bonds from carboxylic acids [32], and has been used for the degradation of bile acid side chains [33], synthesis of BR analogs [14,15,17], and specifically for decarboxylation of the side chain of hyodeoxycholic acid (4) and derivatives [34,35]. Alternatively, the carboxylic degradation reaction may be carried out using PhI(OAc)2/CuSO4 system [13,[34][35][36][37][38][39][40][41][42][43][44][45]. Hyodeoxycholic acid (4) is a common starting material because it is easily available, and it has been previously used to synthesize a number of BR analogs. Related to this work, we have recently reported the synthesis of compound 17 in a five step route with an overall yield of 59% [46]. This compound will be the intermediate for the synthesis of 18 and 19 (Scheme 1).

Synthesis of Brassinosteroids Analogs
Oxidative decarboxylation of the side chain of compound 17, with the Pb(OAc)4/Cu(OAc)2 system, leads to olefin 20 in 75% yield. Formation of 20 was confirmed by 1 H-NMR and 13 C-NMR.

Results
The main goal of this work was to synthesize BR analogs where the main structural change was a reduction of the side alkyl chain length, as compared to brassinolide (1), but keeping the glycol function at the C22−C23 position. In the steroidal nucleus, the introduction of a glycol function at the C22−C23 position via dihydroxylation with OsO 4 requires the presence of a terminal double bond. In the case of hyodeoxycholic acid (4) this can be accomplished by oxidative decarboxylation using a Pb(OAc) 4 /Cu(OAc) 2 system. This method has been proposed to obtain terminal double bonds from carboxylic acids [32], and has been used for the degradation of bile acid side chains [33], synthesis of BR analogs [14,15,17], and specifically for decarboxylation of the side chain of hyodeoxycholic acid (4) and derivatives [34,35]. Alternatively, the carboxylic degradation reaction may be carried out using PhI(OAc) 2 /CuSO 4 system [13,[34][35][36][37][38][39][40][41][42][43][44][45]. Hyodeoxycholic acid (4) is a common starting material because it is easily available, and it has been previously used to synthesize a number of BR analogs. Related to this work, we have recently reported the synthesis of compound 17 in a five step route with an overall yield of 59% [46]. This compound will be the intermediate for the synthesis of 18 and 19 (Scheme 1).

Synthesis of Brassinosteroids Analogs
Oxidative decarboxylation of the side chain of compound 17, with the Pb(OAc) 4 /Cu(OAc) 2 system, leads to olefin 20 in 75% yield. Formation of 20 was confirmed by 1 H-NMR and 13 C-NMR.
The stereochemistry at the C22 carbon for compound 21a was assumed to be 22(S) based on previous results reported for similar hydroxylation reactions used to obtain analogues 10, 13 and 14 ( Figure 2) [15][16][17]. In order to establish the absolute configuration at C22 carbon for compound 21a, the benzoylated derivative 22 (Scheme 1) was prepared. Treatment of 21a with PhCOCl/DMAP in CH2Cl2 and pyridine led to selective esterification of C23 as the only reaction product in 94.0% yield.
Finally, the molecular and crystalline structure of derivative 22 was determined using single crystal X-ray diffraction techniques. This structure crystallizes in the orthorhombic Sohncke space group P212121. The ORTEP diagram appears in Figure 4, whereas X-ray data, bond distances and angles are given in Tables S1-S3 [48]. Nevertheless, the analysis of Bayesian statistics of Bijovet pairs it is a much simpler and reliable method to determine the absolute configuration for molecules that contain atoms no heavier than oxygen [49]. Additionally, considering that compound 22 was synthesized using enantiopure precursors, the configurations R, S, S, S, R, S, S, R and S have also been verified for atoms C3, C5, C8, C9, C10, C13, C14, C17 and C20, respectively.
A mild saponification reaction (K2CO3/MeOH, r.t.) of glycol 21a gave the brassinosteroid analog 9 in 97% yield (Scheme 2). This compound has been previously obtained by using a different synthetic route, and its structure was determined by 1 H-, 13 C-NMR spectroscopy, EIMS and HRMS spectrometry. However, as the assignment of NMR signals was not performed [14] both the 1 H-and 13 C-NMR spectra of this compound are given in the Supplementary Material. Scheme 1. Synthesis of compound 21a, followed by selective benzoylation at the C-23 position to obtain the derivative 22, and synthesis of brassinosteroid analog 9. CC stands for Column Chromatography.
Integration areas of signals in the 1 H-NMR spectrum of mixture 21a/21b, appearing at δ H = 0.965 and 0.928 ppm, respectively, and assigned to the H-21 methyl hydrogen (CH 3 -C20), indicates that the major component on this mixture is the less polar glycol 21a in a ratio 7.5:1.0. Recrystallization of a mixture of 21a/21b (MeOH/Et 2 O = 3/1) allowed for isolation of 21a in 64.0% yield.
The stereochemistry at the C22 carbon for compound 21a was assumed to be 22(S) based on previous results reported for similar hydroxylation reactions used to obtain analogues 10, 13 and 14 ( Figure 2) [15][16][17]. In order to establish the absolute configuration at C22 carbon for compound 21a, the benzoylated derivative 22 (Scheme 1) was prepared. Treatment of 21a with PhCOCl/DMAP in CH 2 Cl 2 and pyridine led to selective esterification of C23 as the only reaction product in 94.0% yield.

Elucidation of Compound Structures
The full structure assignment of compounds 20, 21a, 9, 22, and 23 were carried out by analysis of spectroscopic data obtained from 1 H-NMR, 13 C-NMR, and HRMS of pure and isolated compounds.
In  (Table 1). These data were consistent with those reported for a similar structure but with hydroxyl function at C-3α instead of acetyl group [14,43].
* The 13 C-NMR spectrum of compound 9 was recorded in MeOD solution.
In the 1 H-NMR of 21a signals appearing at chemical shifts δ H = 3.79 ppm, 3.64 ppm, and 3.51 ppm are assigned to protons H-22, H-23a and H-23b, respectively. On the other hand, in the 13 C-NMR spectrum, the signals at δ C = 73.69 and 62.22 ppm correspond to carbinolic carbons C22 and C23, respectively (see Table 1).
The structure of derivative 22 was established mainly by 1D and 2D NMR spectroscopy. In the 1 H-NMR spectrum the aromatic protons appear at δ H = 8.05 (H Ar -2'), 7.58 (H Ar -4'), and 7.46 (H Ar -3') ppm. Additionally, two low field shifted signals at δ H = 4.50 (dd, J = 11.4 and 1.7 Hz, 1H) and 4.20 (dd, J = 11.3 and 4.2 Hz, 1H) ppm correspond to H-23a and H-23b. In the 13 C-NMR spectrum the signal at δ C = 167.01 ppm is assigned to carbonyl of aromatic ester, whereas the signals at δ C = 129.86, 129.63, 128.45 and 133.22 ppm (Table 1) are assigned to aromatic ring (each one of signals at δ C = 129.63 and 128.45 ppm correspond to two symmetrical carbons of the aromatic ring). In the 2D HMBC spectrum a heteronuclear correlation at 3 J H-C between H-23a (δ H = 4.50 ppm) with carbonyl of aromatic ester (δ C = 167.01 ppm) was observed, confirming the presence of benzoyl ester at C23 position. The structure of compounds 18, 19, 24 and 25 were mainly elucidated by analysis of data obtained from 1 H, 13 C, 13 C DEPT-135, 2D HSQC, 2D HMBC NMR, and HRMS measurements.
For compound 24, the position of the 7-oxa lactone function was established from the 1 H-NMR spectrum where a signal observed at δ H = 4.13-4.04 ppm (m, 2H), was assigned to hydrogens H-7 and correlated by 2D 1 H-13 C HSQC with the signal at δ C = 70.31 ppm (CH 2 -7 from 13 C and 13 C DEPT-135 spectra, Table 2). Additionally, important heteronuclear correlations were obtained for hydrogens H-5α and H-7 from a 2D 1 H-13 C HMBC spectrum, i.e., (i) H-7 shows 3 J HC correlations with the signal at δ C = 175.97 ppm (assigned to carbon C-6, C=O of lactone function, Table 2), and signals at δ C = 58.31 ppm (assigned to carbon C-9); and 2 J HC correlation with signal appearing at δ C = 39.42 ppm (assigned to carbon C-8); (ii) H-5α at δ H = 3.03 ppm shows 3 J HC correlation with signals at δ C = 14.54, and 58.31 ppm, which were assigned to carbons CH 3 -19 and C-9, respectively (Table 2); (iii) H-5α exhibits 2 J HC correlation with signals at δ C = 29.7, 36.1 and 176.0 ppm, assigned to carbons C-4, C-10 and C-6, respectively. These correlations are depicted in the 2D HMBC spectrum shown in Figure 5. These observations confirmed unequivocally the 7-oxalactone position for compound 24.  A similar analysis was performed to determine the structure of 6-oxalactone 25. Thus, in the 1 H-NMR spectrum a signal at δH = 4.46 ppm was assigned to H-5α, and correlated by 2D 1 H-13 C HSQC with the signal at δC = 79.68 ppm (CH with impair multiplicity from DEPT-135 spectrum).
Additionally, H-5α shows 2 JHC correlation with signal at δC = 32.95 ppm that is assigned to carbon C-4; and 3 JHC correlation with signals at δC = 11.59, 57.94 and 174.64 ppm, which are assigned to carbons CH3-19, C-9 and C-6, respectively (C=O, of lactone function, Table 2). On the other hand, the 1 H-NMR signal at δH = 2.55-2.43 ppm, corresponding to H-7 (2H, m), shows 2 JHC correlation with signals appearing at δC = 38.31 and 174.64 ppm, which were assigned to carbons C-8 and C-6, respectively ( Table 2); and 3 JHC correlation with signals at δC = 57.97 ppm, assigned to carbons C-9. These correlations are shown in the 2D HMBC spectrum in Figure 6. A similar analysis was performed to determine the structure of 6-oxalactone 25. Thus, in the 1 H-NMR spectrum a signal at δ H = 4.46 ppm was assigned to H-5α, and correlated by 2D 1 H-13 C HSQC with the signal at δ C = 79.68 ppm (CH with impair multiplicity from DEPT-135 spectrum).
Additionally, H-5α shows 2 J HC correlation with signal at δ C = 32.95 ppm that is assigned to carbon C-4; and 3 J HC correlation with signals at δ C = 11.59, 57.94 and 174.64 ppm, which are assigned to carbons CH 3 -19, C-9 and C-6, respectively (C=O, of lactone function, Table 2). On the other hand, the 1 H-NMR signal at δ H = 2.55-2.43 ppm, corresponding to H-7 (2H, m), shows 2 J HC correlation with signals appearing at δ C = 38.31 and 174.64 ppm, which were assigned to carbons C-8 and C-6, respectively (Table 2); and 3 J HC correlation with signals at δ C = 57.97 ppm, assigned to carbons C-9. These correlations are shown in the 2D HMBC spectrum in Figure 6.

General Experimental Methods
All reagents were purchased from commercial suppliers, and used without further purification. Melting points were measured on a SMP3 apparatus (Stuart-Scientific, now Merck KGaA, Darmstadt, Germany) and are uncorrected. 1 H-, 13 C-, 13 C DEPT-135, gs 2D HSQC and gs 2D HMBC NMR spectra were recorded in CDCl 3 or MeOD solutions, and are referenced to the residual peaks of CHCl 3 at δ = 7.26 ppm and δ = 77.00 ppm for 1 H and 13 C, respectively and CD 3 OD at δ = 3.30 ppm and δ = 49.00 ppm for 1 H and 13 C, respectively, on an Avance 400 Digital NMR spectrometer (Bruker, Rheinstetten, Germany) operating at 400.1 MHz for 1 H and 100.6 MHz for 13 C. Chemical shifts are reported in δ ppm and coupling constants (J) are given in Hz, multiplicities are reported as follows: singlet (s), doublet (d), doublet of doublets (dd), doublet of triplets (dt), triplet (t), quartet (q), multiplet (m). IR spectra were recorded as KBr disks in a FT-IR 6700 spectrometer (Nicolet, Thermo Scientific, San Jose, CA, USA) and frequencies are reported in cm −1 . High-resolution mass spectra (HRMS-ESI) were recorded in a Exactive Plus mass spectrometer (Thermo Scientific, Waltham, MA, USA). The analysis for the reaction products was performed with the following relevant parameters: heater temperature, 50 • C; sheath gas flow, 5 (arbitrary unit); sweep gas flow rate, 0 (arbitrary unit) and spray voltage, 3.0 kV at negative mode. The accurate mass measurements were performed at a resolving power: 140,000 FWHM at range m/z 300-500. Optical rotations were measured on a Model AA-5 polarimeter (Optical Activity, Ltd., NJ, USA) with a sodium lamp using a l = 0.1 dm cell and are reported as follows: [α] • C D (c (g/100 mL), solvent). For analytical TLC, silica gel 60 in 0.25 mm layer was used and TLC spots were detected by heating after spraying with 25% H 2 SO 4 in H 2 O. Chromatographic separations were carried out by conventional column on silica gel 60 (230-400 mesh) using EtOAc-hexane gradients of increasing polarity. All organic extracts were dried over anhydrous magnesium sulfate and evaporated under reduced pressure, below 40 • C.

X-ray Crystal Structure Determination
A suitable single crystal of compound 22 was mounted on a MiTeGen MicroMount (MiTeGen, Lansing, NY, USA) in a random orientation. Diffraction data was collected at 120 K on a D8 VENTURE diffractometer (Bruker, Rheinstetten, Germany) equipped with a bidimensional CMOS Photon100 detector, using graphite monochromated Cu-Kα radiation (λ = 1.54178 Å). The diffraction frames were integrated using the APEX2 package. The structure of 22 was solved using Olex2 [51], with the olex2.solve structure solution program using Charge Flipping [52] and refined with full-matrix least-square methods based on F 2 (SHELXL) [53]. Non-hydrogen atoms were refined with anisotropic displacement parameters. All hydrogen atoms were included in their calculated positions, assigned fixed isotropic thermal parameters and constrained to ride on their parent atoms. A summary of the details about crystal data, collection parameters and refinement are documented in Supplementary Material, and additional crystallographic details are in the CIF files. CCDC 1583718 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44-1223-336033; E-mail: deposit@ccdc.cam.ac.uk. ORTEP view was drawn using OLEX2 software [51].

Conclusions
A new synthetic route has been used to obtain the known brassinosteroid analog 9 and new compounds 18, 19, 21a, 22-25. Compound 9 was obtained from 17 in a total yield of 46%, whereas new lactones analogues 18 and 19 were obtained from glycol 21a in 29% and 11% total yields. Additionally, using 1D, 2D NMR, and HRMS we have achieved full structural determination of all compounds shown in Schemes 1 and 2. The absolute stereochemistry at position C-22 was established a (S) by X-ray crystallography studies of the benzoylated derivative 22. This conclusion is in line with literature data reported for similar steroidal structures [14]. Finally, in order to establish a relationship between the side chain structure of BRs analogs and the promoting plant growth activity, additional changes on the side chain should be introduced.